Novel compounds for the treatment of sickle cell disease

ABSTRACT

Compounds have been designed to inhibit the action of cytochrome b5 in the physiological re-reduction of auto-oxidized hemoglobin (methemoglobin), for the purpose of increasing methemoglobin levels in the blood of patients as a treatment for sickle cell disease. Administration of the compounds mimics congenital deficiencies in cytochrome b5, in which methemoglobin levels rise as high as 50% of total hemoglobin and derivatives in the blood, without adverse clinical manifestations. Methemoglobin inhibits red cell sickling and high levels of methemoglobin in the blood induced by the compounds of this invention prevent the symptoms of sickle cell disease.

This application claims the benefit of U.S. Provisional Application No.60/443,783, filed Jan. 30, 2003, incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to compounds that bind to and inhibit theactivity of cytochrome b₅ in the physiological re-reduction ofauto-oxidized hemoglobin (methemoglobin), e.g., compounds that interferewith the binding of hemoglobin and/or methemoglobin to cytochrome b₅.The invention further relates to pharmaceutical compositions comprisingthese compounds, and methods of using these pharmaceutical compositionsto increase methemoglobin levels in the blood as a treatment for sicklecell disease.

2. Description of the Related Art

In the United States the occurrence of sickle cell disease, also knownas sickle cell anemia, is relatively low, afflicting only about 70,000Americans (Jones, C. P. 1995, Pharmacy Today, 1, 1-2), predominantly ofAfrican descent. Worldwide, however, sickle cell disease afflicts manymillions of individuals (Bunn, H. F.; Forget, B. G. Hemoglobin:Molecular, Genetic and Clinical Aspects; W. B. Saunders Company:Philadelphia, 1986. 502-564). Despite the fact that the molecularmechanism of hemoglobin sickling is well understood (Eaton, W. A.;Hofrichter, J. 1990, Adv. Protein Chem., 40, 63-279) and the role ofsickling in the pathology of the disease is clear, a rationally baseddrug therapy has not heretofor been available to patients despite manyattempts to develop such a therapy over the last few decades (Orringer,E. P.; Casella, J. P.; Ataga, K. I.; Koshy, M.; Adams-Graves, P.;Luchtman-Jones. L.; Wun, T.; Watanabe, M.; Shafer, F.; Kutlar, A.;Abboud, M.; Steinberg, M.; Adler, B.; Swerdlow, P.; Terregino, C.;Saccente, S.; Files, B.; Ballas, S.; Brown, R.; Wojtowicz-Praga, S.;Grindel, J. M. 2001, JAMA, 286(17):2099; Abraham, D. J.; Perutz, M. F.;Phillips, S. B. 1983, Proc. Natl. Acad. Sci. USA, 80, 324-328; Klotz, I.M.; Haney, D. N.; King, L. C. 1981 Science,213, 724-731). Althoughagents that modify hemoglobin allosterism have been identified throughsite-directed drug design (Abraham, D. J.; Wireko, F. C.; Randad, R. S.;Poyart, C.; Kister, J.; Bohn, B.; Liard, J. F.; Kunert, M. P. 1992,Biochemistry, 31, 9141-9149), to date, attempts at hemoglobin-directedantisickling agents have been unsuccessful.

The clinical manifestations of sickle cell disease are highly variable(Bunn, H. F.; Forget, B. G. Hemoglobin: Molecular, Genetic and ClinicalAspects; W. B. Saunders Company: Philadelphia, 1986. 502-564; Serjeant,G. R. 2001, Br. J. Haematol., 112, 3-18). In young children, the majorconcern is the incidence of stroke. Chronic hemolytic anemia, impairmentof growth and higher susceptibility to infection are common systemicmanifestations. Vaso-occlusive crises are the origin of the most severesymptoms, including stroke and cardiac involvement in cases of “chestsyndrome”, The incidence of three or more vaso-occlusive crises per yearis highly correlated with mortality. With current treatment, lifeexpectancy for sickle cell patients is 40 to 50 years.

Until recently, the only approach to the treatment of sickle celldisease was fluids and analgesics, such as morphine, administered uponthe occurrence of vaso-occlusive crises.

While transfusion therapy is commonly employed in pediatric cases wherethe risk of stroke is high, there are serious potential problems withlong-term transfusion therapy (Serjeant, G. R. 2001, Br. J. Haematol.,112, 3-18). The risk of stroke is predicted by transcranial dopplermeasurements of blood flow in the brain. Although transfusion therapy iseffective in reducing vaso-occlusive crises, such as stroke, there areseveral drawbacks. Iron overload is a common side effect, and ironchelation therapy employing desferoxamine is a common adjuvant therapy.Long-term transfusion therapy also carries the risk of alloimmunogenicreactions. There is also a risk of disease transmission that has beenminimized with recent advances in diagnostic procedures.

In 1995, hydroxyurea (Jones, C. P. 1995 Siclde Cell Therapy soEffective, Trials end early, Pharmacy Today, 1, 1-2; Charache, S.;Terrin, M. L.; Moore, R. D.; Dover, G. J.; Barton, F. B.; Eckert, S. V.;McMahon, R. P.; Bonds, D. R. 1995, N. Engl. J. Med., 332, 317-1322;Rodgers, G. P. 1997 Semin. Hematol., 34, 2-7.) became available for thetreatment of sickle cell disease. Because it was already used in thetreatment of certain leukemias, it was rapidly approved for clinicaltesting and passed through clinical trials faster than any other drug inrecent times. In an extensive study involving nearly 300 sickle cellpatients, the occurrence of vaso-occlusive crises was reduced to roughly50% of that observed in the patients involved (Charache, S.; Terrin, M.L.; Moore, R. D.; Dover, G. J.; Barton, F. B.; Eckert, S. V.; McMahon,R. P.; Bonds, D. R. 1995, N. Engl. J. Med., 332, 317-1322.). It isbelieved that hydroxyurea works by inducing expression of fetalhemoglobin, however, there are a number of controversies concerning theexact mechanism of action, given that benefits appear to begin beforethe development of significant levels of fetal hemoglobin (Bunn, H. F.1999, Blood, 93, 1787-1789). Despite the advantages in the use ofhydroxyurea, it is not without significant side effects. Hydroxyurea ismyelosuppressive and thus patients must be monitored carefully (Rodgers,G. P. 1997 Semin. Hematol., 34, 2-7). Hydroxyurea causes chromosomalfragmentation and is teratogenic and mutagenic but does not appear to becarcinogenic. Because of the mutagenicity and the potentialcarcinogenicity in the long-term, it is not approved for use inchildren. Rather, it is approved for use in patients who suffer morethan three vaso-occlusive crises a year, a clinical pattern stronglycorrelated with mortality (Castro, O. 1999, Br. J. Haematol., 107,2-11).

Relatively recently, bone marrow transplantation has been found to be aneffective cure for sickle cell disease (Serjeant, G. R. 2001, Br. J.Haematol., 112, 3-18). This treatment was first discovered when aleukemia patient was given a bone marrow transplant and serendipitouslywas also cured of his sickle cell disease. Several hundred bone marrowtransplants have been performed specifically for the purpose of treatingsickle cell disease. This approach is only available to about 18% ofsickle cell patients because of the requirement of an HLA matchedsibling donor. The procedure is costly and carries significant risks.Mortality because of immune responses ranges from 10% to 15% andsubsequent alloimmune responses can be problematic. Thus, although bonemarrow transplantation is a very promising cure for the geneticdisorder, it has significant limitations that prevent widespread use.

It is known that methemoglobin, oxyhemoglobin, andcarbonmonoxyhemoglobin, effectively inhibit sickling in patients withsiclde cell disease (Franklin, I. M.; Rosemeyer, M. A.; Huehns, E. R.1983, Br. J. Haematol., 54, 579-587). Furthermore, in individuals withcongenital deficiencies in cytochrome b₅, methemoglobin levels rise ashigh as 50% of total hemoglobin and derivatives in the blood, withoutany adverse clinical manifestations other than mild cyanosis. Clinicaltrials performed in the 1960's demonstrated the efficacy ofmethemoglobin in the suppression of vaso-occlusive crises but werelimited by the rapid re-reduction of methemoglobin by cytochrome b₅ andhence required massive quantities of compounds such as sodium nitrite,benzocaine or para-aminopropriophenone to maintain sufficient levels ofmethemoglobin (Beutler, E. 1961, J. Clin. Invest., 40, 56-68). Deliveryof the appropriate quantities of these compounds was difficult and theprospect of good patient compliance with such a drug regimen was remote.Thus, sickle cell disease is a serious problem for which no effectivesolution is available, and a potentially useful approach to thetreatment of the disease would be to increase the amount ofmethemoglobin in patients having siclde cell disease.

Cytochrome b₅ is the terminal electron donor to methemoglobin in thephysiological re-reduction of auto-oxidized hemoglobin (Abe, K.; Sugita,Y. 1979, Eur. J. Biochein., 101, 423-428; Gerbaut, L. 1991 Clin. Chem.,37, 2117-2120). Hemoglobin auto-oxidizes at approximately 3% per day.The structures for hemoglobin and its derivatives have been previouslydetermined (Perutz, M. F. 1989 TIBS, 14, 42-44; Bolton, W.; Cox, J. M.;Perutz, M. F. 1968, J Mol Biol, 33, 283-297) and as well as that ofcytochrome b₅ (Mathews, S.; Czerwinski, E. W.; Argos, P. The X-RayCrystallographic Structure of Calf Liver Cytochrome b ₅; Dolphin, D.,Ed.; Academic Press: New York, 1979; Vol. VII, pp 107-147). Complete NMRassignments for the rat cytochrome b₅ were determined for bothequilibrium forms (Guiles, R. D.; Basus, V. J.; Kuntz, I. D.; Waskell,L. 1992, Biochemistry, 31, 11365-11375; Guiles, R. D.; Basus, V. J.;Sarma, S.; Malpure, S.; Fox, K. M.; Kuntz, I. D.; Waskell, L. 1993,Biochemistry, 32, 8329-8340). In addition, extensive characterization ofthe structural, dynamic, and electrochemical properties of ratcytochrome b₅ have been performed (Dangi, B.; Sarma, S.; Yan, C.;Banville, D. L.; DiGate, R. J.; Guiles, R. D. 1998, Biochemistry,37,8289-830251-54; Dangi, B.; Blankman, J. I.; Miller, C. J.; Volkman, B.F.; Guiles, R. D. 1998, J. Phys. Chem B,102, 8201-8208; Sarma, S.;Banville, D.; DiGate, R. J.; Miller, C.; Guiles, R. D. 1997,Biochemistry,36, 5658-5668; Cheng, J.; Terrettaz, S.; Blankman, J. I.;Miller, C. J.; Dangi, B.; Guiles, R. D. 1997, Israel Journal ofChemistry,37, 259-266; Blankman, J. I.; Shahzad, N.; Dangi, B.; Miller,C. J.; Guiles, R. D. 2000, Biochemistry,39, 14799-14805). Furthermore,theoretical studies of the hemoglobin and cytochrome b₅ complex havebeen performed (Poulos, T. L.; Mauk, A. G. 1983, J. Biol. Chem., 258,7369-7373).

SUMMARY OF THE INVENTION

The present invention is directed to compounds that inhibit cytochromeb₅'s action in the re-reduction of methemoglobin to hemoglobin, whichthereby leads to an increase in methemoglobin levels. Thus, thecompounds of the present invention are useful in treating sickle celldisease.

According to a preferred embodiment, the present invention provides acompound of the formula R1-R2-R3, wherein: R1 comprises a moiety thatbinds to the hemoglobin binding site on cytochrome b₅ and competitivelyinhibits hemoglobin binding to cytochrome b₅; R3 comprises a moiety thatbinds to cytochrome b₅ at a site distinct from the site at which R1binds to cytochrome b₅; and R2 comprises a moiety that links R1 and R3.

In one preferred embodiment of a compound of the present invention, R1is a linear polyamine.

In another preferred embodiment of a compound of the present invention,R1 is a cyclic polyamine.

In another preferred embodiment of a compound of the present invention,R1 is a hexacyclen.

In another preferred embodiment of a compound of the present invention,R1 is a moiety that binds to cytochrome b₅ at one or more amino acidsselected from the group consisting of H26, E43, E44, E48, A54, D60, H80and A88.

In another preferred embodiment of a compound of the present invention,R3 is a moiety that binds to the ATP binding site on cytochrome b₅.

In another preferred embodiment of a compound of the present invention,R3 is ATP or an ATP analog.

In another preferred embodiment of a compound of the present invention,R3 is β-nicotinamide adenine dinucleotide.

In another preferred embodiment of a compound of the present invention,R3 is ATP; 1,N6-ethenoadenosine 5′ triphosphate; β-nicotinamide adeninedinucleotide; 1,N6-ethenoadenosine hydrochloride;nicotinamide-1,6-ethenoadenosine; or coenzyme A.

In another preferred embodiment of a compound of the present invention,R3 is a moiety that binds to cytochrome b₅ at one or more amino acidsselected from the group consisting of I24, L25, H26 and H27.

In a particular preferred embodiment of a compound of the invention, R1is hexacyclen and R3 is β-nicotinamide adenine dinucleotide.

In one preferred embodiment of a compound of the present invention, R2is a flexible linker.

In another preferred embodiment of a compound of the present invention,R2 is a moiety that covalently crosslinks R1 and R3.

In another preferred embodiment of a compound of the present invention,R2 is a polyglycine moiety.

In another preferred embodiment of a compound of the present invention,R2 is polyethylene glycol (PEG); polystyrene-PEG;[2-(2-aminoethoxy)ethoxy] acetic acid;allyloxycarbonyl-[2-(2-aminoethoxy)ethoxy] acetic acid;fluorenyl-methoxycarbonyl-[2-(2-aminoethoxy)ethoxy] acetic acid;ter-butyloxycarbonyl-[2-(2-aminoethoxy)ethoxy] acetic acid;benzyloxycarbonyl-[2-(2-aminoethoxy)ethoxy] acetic acid; or BMPS(N-(β-maleimido-propyloxy)succinimide).

In another preferred embodiment of a compound of the present invention,R2 is a straight chain or branched chain hydrocarbon.

In a preferred embodiment of a compound of the present invention, saidcompound binds to cytochrome b₅ and inhibits the activity of cytochromeb₅ in the reduction of methemoglobin to hemoglobin.

According to another preferred embodiment, the present inventionprovides a pharmaceutical composition comprising a compound of thepresent invention or a pharmaceutically acceptable salt thereof.

According to another preferred embodiment, the present inventionprovides a method of reducing the incidence of red blood cell sicklingin a patient with sickle cell disease, comprising administering aneffective amount of a compound of the present invention to the patient.

According to another preferred embodiment, the present inventionprovides a method of raising the level of methemoglobin in blood,comprising adding an effective amount of a compound of the invention tothe blood. In one embodiment of this method, the compound is added tothe blood ex vivo.

According to another preferred embodiment, the present inventionprovides a method of raising the level of methemoglobin in the blood ofa patient, comprising administering an effective amount of a compound ofthe present invention to the patient.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B illustrate sections of contour plots of overlays of¹H-¹⁵N HSQC spectra of cytochrome b₅ by itself and in complex with humanmethemoglobin. In FIG. 1A, the concentration of cytochrome b₅ is 1 mMand the concentration of methemoglobin is 0.50 mM. In FIG. 1B, theconcentration of methemoglobin is 0.25 mM.

FIG. 2 illustrates the heteronuclear correlation spectra (HSQC spectra)of a 2 mM solution of cytochrome b₅ by itself (black contours) and thatof a solution containing 2 mM cytochrome b₅ and 4 mM hexacyclen (graycontours).

FIG. 3 shows modification of hexacyclen to enable attachment of an R2linker for use in crosslinking to the R3 moiety.

FIG. 4 shows the thiolation of ADP that can then be linked to thederivatized polyamines.

FIG. 5 shows linking of derivatized spermine to derivatized ADP.

FIG. 6 shows attachment of the flexible spacer and covalent attachmentof the two derivatized groups.

FIG. 7 shows an HSQC overlay of a sample containing cytochrome b₅ andATP (2 mM) and a sample of cytochrome b₅ alone.

FIG. 8 shows a set of traces of the optical absorbance changes occurringat 577 nm for cytochrome b₅ and methemoglobin at 5 μM concentrations.Various concentrations of buffer and of hexacyclen were examined. Intrace A) the buffer concentration is 10 mM phosphate at pH 7.0. In traceB) the buffer concentration is 1 mM phosphate at pH 7.0. In trace C) thebuffer concentration is 1.0 mM phosphate pH 7.0 and the concentration ofhexacyclen is 100 μM. In trace D and E) the concentration of phosphateis 1 mM pH 7.0 and the concentration of hexacyclen is 1 mM.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compounds having the structure R1-R2-R3that bind to cytochrome b₅ and inhibit the activity of cytochrome b₅ inthe reduction of methemoglobin to hemoglobin. Without wishing to bebound to any particular mechanism, it is proposed that these compoundsprevent the binding of hemoglobin and/or methemoglobin to cytochrome b₅by binding with high affinity to the methemoglobin/hemoglobin bindingsite on cytochrome b₅ and preventing the electron transfer betweenmethemoglobin/hemoglobin and cytochrome b₅. By binding to cytochrome b₅and preventing reduction of autoxidized hemoglobin, these compoundsraise the level of methemoglobin in red blood cells and reduce theincidence of cell sickling. Thus, these compounds are useful for thetreatment of sickle cell disease.

In one embodiment, the invention relates to a compound that comprisesthree parts, designated R1, R2 and R3 as described below. R1 and R3 bindto specific sites on the surface of cytochrome b₅ as defined by shiftsin ¹H-¹⁵N heteronuclear correlation spectrum peaks defined below(Heteronuclear Single Quantum Coherence (HSQC) mapping: Mori, S.;Abeygunawardana, C.; Johnson, M. 0. N.; van Zijl, P. C. M. 1995, Journalof Magnetic Resonance, Series B, 108, 94-98). R2 is a linker whichcovalently links R1 and R3.

R1 is a moiety that binds to specific sites on cytochrome b₅ in a waythat mimics hemoglobin binding to cytochrome b₅, except preferably withhigher affinity than hemoglobin binding. Thus, R1 competitively inhibithemoglobin binding to cytochrome b₅. Furthermore, the high affinity ofR1 to the hemoglobin binding site of cytochrome b₅ inhibits electrontransfer from cytochrome b₅ to methemoglobin.

One can use an optical assay of electron transfer rate to identify suchmoieties (see examples below). It is believed that moieties thatinterfere with electron transfer between cytochrome b₅ and methemoglobinalso bind to one or more of the following cytochrome b₅ residues: H26,E43, E44, E48, A54, D60, H80 and A88, as shown by shifts in HSQCperturbation mapping (see examples below). (Shuker, S. B.; Hajduk, P.J.; Meadows, R. P.; Fesik, S. W. 1996, Science, 274, 1531-4).

In a preferred embodiment, R1 is 1,4,7,10,13,16-hexaazacylooctadecane(hexacyclen), the structure of which is as follows:

In other preferred embodiments, R1 is a derivative of hexacyclen, suchas that described in Example 4.

R2 is a linker between R1 and R3. Although a number of linkers, flexibleand non-flexible, are known in the art field, it is preferable that thelinker, R2, be flexible. In one embodiment, R2 is a polyglycine moietycontaining between 1 and 3 glycines. In another embodiment, R2 ispolyethylene glycol (PEG), or a PEG-like moiety such as, but not limitedto, polystyrene-PEG, [2-(2-aminoethoxy)ethoxy] acetic acid,allyloxycarbonyl-[2-(2-aminoethoxy)ethoxy] acetic acid,fluorenyl-methoxycarbonyl-[2-(2-aminoethoxy)ethoxy] acetic acid,ter-butyloxycarbonyl-[2-(2-aminoethoxy)ethoxy] acetic acid, orbenzyloxycarbonyl-[2-(2-aminoethoxy)ethoxy] acetic acid. In stilladditional embodiments, R2 is a straight chain or branched chain ofcarbon and hydrogen where the number of carbon atoms is 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, or more. An appropriate-lengthlinker connects R1 and R3 when they are both bound to their respectivebinding sites on cytochrome b₅.

R3 is a moiety that binds to a site on cytochrome b₅ distinct from thebinding site of R1. Preferably, R3 binds to specific sites on cytochromeb₅ in a way that mimics ATP binding to cytochrome b₅. This additionaland distinct binding of R3 to cytochrome b₅ increases the overallaffinity of a compound of the present invention for cytochrome b₅. Thus,even at the relatively high salt concentration of blood, compounds ofthe present invention bind with high affinity to cytochrome b₅.

It is preferable that R3 binds to cytochrome b₅ and induces shifts inheteronuclear correlation peaks corresponding to one or more offollowing residues on cytochrome b₅: I24, L25, H26, and H27. In apreferred embodiment of the invention, R3 is ATP (adenosine5′-triphosphate); 1,N6-ethenoadenosine 5′ triphosphate; β-nicotinamideadenine dinucleotide; 1,N6-ethenoadenosine hydrochloride;nicotinamide-1,6-ethenoadenosine; or coenzyme A. In another preferredembodiment of the invention, R3 is any ATP analog, many of which existand are well known in the art-field. ATP binds to cytochrome b₅ with anaffinity of 180 μM. Below are the chemical structures of some R3moieties according to preferred embodiments of the invention, all ofwhich are commercially available:

Adenosine 5′-triphosphate (ATP);

1,N6-Ethenoadenosine 5′ triphosphate;

β-Nicotinamide adenine dinucleotide;

1,N6-ethenoadenosine hydrochloride;

Nicotinamide-1,6-ethenoadenosine;

Coenzyme A.

The present invention further provides a method of reducing theincidence of red blood cell sickling in a patient with sickle celldisease and in need of treatment thereof, comprising administering aneffective amount of a compound according to the present invention to thepatient.

This invention also provides a method for preventing the reduction ofmethemoglobin to hemoglobin such that methemoglobin accumulates in theblood, comprising administering an effective amount of a compoundaccording to the present invention. Such an accumulation ofmethemoglobin is useful for the prevention of sickling events inpatients having sickle cell disease. Thus, the present inventionprovides a method of raising the level of methemoglobin in the blood ofa patient, comprising administering an effective amount of a compoundaccording to the present invention to the patient.

The invention also provides a method of raising the level ofmethemoglobin in blood, comprising adding an effective amount of acompound of the invention to the blood. For example, the compound canadded to the blood ex vivo. This blood can then be used to transfuse apatient having sickle cell disease.

Cytochrome b₅ plays an important role in reducing methemoglobin levels,as demonstrated experimentally and indicated in individuals withcongenital deficiencies in cytochrome b₅, who have abnormally highlevels of methemoglobin in their blood. Compounds of the invention caninhibit the activity of cytochrome b₅ by, e.g., blocking the binding ofmethemoglobin to cytochrome b₅. Compounds of the invention can alsoachieve the inhibition of cytochrome b₅ activity by, e.g., blockingelectron transfer to methemoglobin. Compounds of the invention that arecomprised of two moieties such that each moiety binds to different siteson cytochrome b₅ have an affinity for cytochrome b₅ that is greater thanthe affinity of either moiety for its individual site. The compounds ofthe invention are thus highly effective at inhibiting cytochrome b₅activity and raising levels of methemoglobin in the blood.

The inventive compounds exhibit therapeutic activity in raising levelsof methemoglobin in the blood, and are effective in treating sickle celldisease by reducing the amount of cell sickling. In accordance with apreferred embodiment, the present invention includes methods of treatingpatients suffering from sickle cell disease.

The present invention further provides pharmaceutical compositionscomprising a compound according to the present invention and apharmaceutically acceptable carrier; a method of inhibiting the activityof cytochrome b₅ in red blood cells by administering a pharmaceuticalcomposition of the invention to a patient; a method of increasing thelevels of methemoglobin in red blood cells by administering apharmaceutical composition of the invention to a patient; and a methodof treating sickle cell disease in a patient by administering apharmaceutical composition of the invention to the patient.

The present invention also relates to useful forms of the compounds asdisclosed herein, such as pharmaceutically acceptable salts and prodrugsof all the compounds. The compounds of the invention can be administeredalone or as an active ingredient of a formulation. Thus, the presentinvention also includes pharmaceutical compositions of compounds of theinvention containing, for example, one or more pharmaceuticallyacceptable carriers.

Numerous standard references are available that describe procedures forpreparing various formulations suitable for administering the compoundsaccording to the invention. Examples of potential formulations andpreparations are contained, for example, in the Handbook ofPharmaceutical Excipients, American Pharmaceutical Association (currentedition); Pharmaceutical Dosage Forms: Tablets (Lieberman, Lachman andSchwartz, editors) current edition, published by Marcel Dekker, Inc., aswell as Remington's Pharmaceutical Sciences (Arthur Isol, editor),1553-1593 (current edition).

In view of the high degree of selective inhibition of cytochrome b₅activity, the compounds of the present invention can be administered toa patient requiring inhibition of cytochrome b₅ activity. Administrationmay be accomplished according to patient's needs, for example, byintravenous injection. Various solid oral dosage forms can be used foradministering compounds of the invention including such solid forms astablets, gelcaps, capsules, caplets, granules, lozenges and bulkpowders. The compounds of the present invention can be administeredalone or combined with various pharmaceutically acceptable carriers,diluents (such as sucrose, mannitol, lactose, starches) and excipientsknown in the art, including but not limited to suspending agents,solubilizers, buffering agents, binders, disintegrants, preservatives,colorants, flavorants, lubricants and the like. Time-release capsules,tablets and gels are also advantageous in administering the compounds ofthe present invention.

Various liquid oral dosage forms can also be used for administeringcompounds of the inventions, including aqueous and non-aqueoussolutions, emulsions, suspensions, syrups, and elixirs. Such dosageforms can also contain suitable inert diluents known in the art such aswater and suitable excipients known in the art such as preservatives,wetting agents, sweeteners, flavorants, as well as agents foremulsifying and/or suspending the compounds of the invention. Thecompounds of the present invention may be injected, for example,intravenously, in the form of an isotonic sterile solution. Otherpreparations are also possible.

The compounds can be administered as the sole active agent or incombination with other pharmaceutical agents, such as other agents thatraise levels of hemoglobin variants in the red blood cells in order toprevent cell sickling in patients with sickle cell disease.

The dosages of the compounds of the present invention depend upon avariety of factors including the severity of the symptoms, the age, sexand physical condition of the patient, the route of administration, thefrequency of the dosage interval, the particular compound utilized, theefficacy, toxicology profile, pharmacokinetic profile of the compound,and the presence of any deleterious side-effects, among otherconsiderations.

By “effective dose” or “therapeutically effective dose” or “effectiveamount” is meant herein, in reference to the treatment of sickle celldisease, an amount sufficient to bring about one or more of thefollowing results: increase the level of methemoglobin in the bloodabove about 3%; reduce the level of pain related to sickle cell disease;or reduce the incidence of sickle cell crises. The compounds of theinvention can be administered at dosage levels and in a manner customaryfor ticlopidine hydrochloride, or other drugs used to treat sickle celldisease. For example, ticlopidine hydrochloride is administered at 250mg bi-daily (see Physicians' Desk Reference, the relevant portion ofwhich incorporated herein by reference). However, the concentration ofcytochrome b₅ in blood is 5000 times lower than the concentration ofhemoglobin (0.2 μM compared to 1 mM). Therefore, a compound of theinvention targeted to cytochrome b₅ could potentially be administered ata dose of up to 5000 times lower than the dose of a sickle cell drugthat is targeted to hemoglobin, e.g. ticlopidine hydrochloride. By thisextrapolation, a compound of the invention could be administered at adose of only 50 μg twice daily.

In carrying out the procedures of the present invention it is of courseto be understood that reference to particular buffers, media, reagents,cells, culture conditions and the like are not intended to be limiting,but are to be read so as to include all related materials that one ofordinary skill in the art would recognize as being of interest or valuein the particular context in which that discussion is presented. Forexample, it is often possible to substitute one buffer system or culturemedium for another and still achieve similar, if not identical, results.Those of skill in the art will have sufficient knowledge of such systemsand methodologies so as to be able, without undue experimentation, tomake such substitutions as will optimally serve their purposes in usingthe methods and procedures disclosed herein.

EXAMPLES Example 1

The interaction of cytochrome b₅ with hemoglobin is explored using HSQCperturbation mapping. FIG. 1 contains sections of contour plots ofoverlays of ¹H-¹⁵N HSQC spectra of cytochrome b₅ by itself and incomplex with human methemoglobin. Significant shifts in the positions ofa number of residues of cytochrome b₅ are observed. In FIG. 1A, theconcentration of cytochrome b₅ is 1 mM and the concentration ofmethemoglobin is 0.50 mM. The black contours are of a heteronuclearcorrelation spectrum of cytochrome b₅ by itself while the gray contoursare of a sample containing both cytochrome b₅ and methemoglobin. In FIG.1B, the concentration of methemoglobin is 0.25 mM. The pH in all caseswas 6.4 and the temperature was 25° C. A number of residues that shiftsignificantly on complex formation are labeled (e.g. most notably H26,E43, E44, A54, H80 and A88). A number of peaks that do not shiftsignificantly on complex formation are also labeled (i.e. K5 and Y30).Heteronuclear correlation spectra were recorded using the fast HSQCsequence (Mori, S.; Abeygunawardana, C.; Johnson, M. O. N.; van Zijl, P.C. M. 1995, J. Mag. Reson. B, 108, 94-98). The shifts in heteronuclearcorrelation peaks observed on complex formation are consistent at leastin part with the theoretical model of the complex between cytochrome b₅and methemoglobin. The shifts in peaks associated with residues E43, E44and probably A54 via a relayed effect in helix V of cytochrome b₅ areconsistent with the theoretical model. Compounds which interact withcytochrome b₅ at one or more of the following amino acids of cytochromeb₅ are proposed to interfere with or prevent the binding ofmethemoglobin to cytochrome b₅, the amino acids being H26, E43, E44,A54, H80 and A88.

Example 2

Hexacyclen (1,4,7,10,13,16-hexaazacyclooctadecane)(Richman, J. E.;Atkins, T. J. 1974, J. Am. Chem. Soc., 96, 2268-2269) binds tocytochrome b₅ such the HSQC spectra of cytochrome b₅-hexacyclen issimilar to the HSQC spectra of cytochrome b₅-hemoglobin. Theconcentration dependence of hexacyclen-induced heteronuclear correlationpeak shifts indicates a dissociation constant of roughly 2 mM. FIG. 2illustrates the heteronuclear correlation spectra (HSQC spectra) of a 2mM solution of cytochrome b₅ by itself (black contours) and that of asolution containing 2 mM cytochrome b₅ and 4 mM hexacyclen (graycontours). The inset in the upper left hand corner of the figurecontains a plot of the hexacyclen dependence of the shifts in the peakto peak separation of aspartate 60 (D60) at concentrations of hexacylenranging from 0.5 to 8 mM. The inset at the upper right is a model forthe interaction of hexacyclen based on the shifts observed in the HSQCperturbation study. Solutions were buffered to a pH of 7.0 with 1 mMphosphate buffer and the spectra were recorded at 40° C. The insert inthe upper left hand corner of the figure contains a plot of thehexacyclen dependence of the shifts in the peak to peak separation ofaspartate 60 (D60) at concentrations of hexacylen ranging from 0.5 to 8mM. The insert at the upper right is a model for the interaction ofhexacyclen based on the shifts observed in the HSQC perturbation study.

Example 3

Work has been performed on cytochrome b₅ in an attempt to find compoundswhich inhibit or increase cytochrome b₅'s properties, such as thereduction potential and binding affinity to cytochrome b₅'s electrontransfer partners by relayed effects (Rivera, M.; Wells, M. A.; Walker,F. A. 1994, Biochemistry, 33, 2161-2170; Vergeres, G.; Waskell, L. 1995,Biochemie, 77, 604-620; Reid, L. S.; Gray, H. B.; Dalvit, C.; Wright, P.E.; Saltman, P. 1987, Biochem., 26, 7102-7107). ATP binds to cytochromeb₅ with an affinity of 180 μM (Reid, L. S.; Gray, H. B.; Dalvit, C.;Wright, P. E.; Saltman, P. 1987, Biochem., 26, 7102-7107). ATP binds toa hydrophobic domain between the four helix bundle that binds the hemeand the β-turn in the β-sheet region on cytochrome b₅, this binding sitebeing distinct from the binding site of hexacyclen.

Example 4

Schemes for the derivatization of hexacyclen (R1) have been developedusing minor modifications of the Richman-Atkins synthesis(Richman, J.E.; Atkins, T. J. 1974, J. Am. Chem. Soc., 96, 2268-2269). The R groupin the scheme shown in FIG. 3 was a tosyl group in the originalsynthesis, but has been replaced with a carbamyl group in thismodification, which can be selectively removed using L-selectride (Coop,A.; Rice, K. C. 1998, Tet. Lett., 39, 8933-8934), lithiumtri-sec-butylborohydride (Aldrich), under mild conditions which will notremove the Ts group. FIG. 3 shows a modified Richman-Atkins synthesis ofhexacyclen to enable attachment of a derivatizable R group for use incrosslinking to the R3 moeity. A scheme utilizing this modifiedhexacyclen in the preparation of a polypeptide that is used to link thethiolated ADP shown in FIG. 4 using a bifunctional crosslinking agent isshown in FIG. 6.

Example 5

In this scheme to link hexacyclen (R1) with an ADP derivative (R3),described by Hermanson (Hermanson, G. T. Bioconjugate Techniques;Academic Press, 1995.785 pp649-655), a water soluble carbodiamide isused to activate the terminal phosphate which can then be thiolated withβ-mercaptoethylamine via cystamine following reductive cleavage withCleland's reagent as shown in FIG. 4. As shown in FIG. 4, a watersoluble carbodiamide is used to activate the terminal phosphate that canthen be thiolated with β-mercaptoethylamine via cystamine followingreductive cleavage with Cleland's reagent.

Example 6

In this scheme to link hexacyclen (R1) with an ADP derivative (R3), apolypeptide containing a C-terminal lysine and N-termination with thederivatized spermine is prepared using standard solid phase methods(Grant, G. A. Synthetic Peptides: A User's Guide; W. H. Freeman andCompany: New York, 1992, 382). This polypeptide is then crosslinked tothe derivatized ADP shown in FIG. 3 using the bifunctional crosslinkerBMPS (N-(β-maleimido-propyloxy)succinimide) (McKenzie, J. A.; Raison, R.I.; Rivett, E. E. 1988, J. Protein Chem., 7, 581-592) as shown in FIG.5.

Example 7

A similar scheme for linking a polypeptide with an N-terminal hexacylenderivative to the thiolated ADP is shown in FIG. 6. In this scheme apolypeptide with a C-terminal lysine and a variable number of glycinesis prepared using standard solid phase synthesis techniques. TheN-terminal group here is the derivatized hexacyclen.

Example 8

The binding of the R1 and R3 moieties to cytochrome b₅ is characterizedusing HSQC perturbation mapping experiments. FIG. 2 contains an overlayof heteronuclear correlation spectra (HSQC spectra) of a 2 mM solutionof cytochrome b₅ by itself (black contours) and that of a solutioncontaining 2 mM cytochrome b₅ and 4 mM hexacyclen (gray contours),illustrating shifts due to the binding of hexacyclen to ¹⁵N-labeledcytochrome b₅. FIG. 2 can be compared with FIG. 7, which contains anHSQC overlay of a control on that of a sample containing cytochrome b₅and ATP. Solutions were buffered to a pH of 7.0 with 1 mM phosphatebuffer and the spectra were recorded at 40° C. FIG. 7 contains an HSQCoverlay of a control on that of a sample containing cytochrome b₅ andATP at 2 mM concentration. Although there is some overlap in peaksaffected by the binding of ATP with that seen with the binding ofhexacyclen, some of these effects are probably relayed.

Example 9

In addition to the. site interaction studies using NMR, functionalassays of inhibition of electron transfer have been performed usingmanual mixing experiments, similar to those described by Sugita (Abe,K.; Sugita, Y. 1979, Eur. J. Biochem. 101, 423-428). The electrontransfer reactions were monitored by observing absorbance changes at 577nm similar to experiments performed by McLendon's group (Qiao, T.;Simmons, J.; Horn, D. A.; Chandler, R.; McLendon, G. 1993, 97,13089-13091). FIG. 8 contains a set of traces of the optical absorbancechanges occurring at 577 nm for cytochrome b₅ and methemoglobin at 5 μMconcentrations with 1 mM phosphate buffer at pH 7.0. Variousconcentrations of hexacyclen were examined ranging from 100 μM to 1 mM.The concentration of phosphate buffer was also examined in order toassess the effect of ionic strength on the rate of the reaction. In allcases the concentration of cytochrome b₅ and methemoglobin is 5 μM andthe temperature was maintained at 37° C. For trace A, the bufferconcentration is 10 mM phosphate at pH 7.0. For trace B, the bufferconcentration is 1 mM phosphate at pH 7.0. For trace C, the bufferconcentration is 1.0 mM phosphate, pH 7.0 and the concentration ofhexacyclen is 100 mM. For trace D and trace E, the concentration ofphosphate is 1 mM, pH 7.0 and the concentration of hexacyclen is 1 mM.

While the disclosure above describes the invention in detail and withreference to specific embodiments thereof, it will be apparent to one ofordinary skill in the art that various changes and modifications can bemade therein without departing from the spirit and scope thereof.

1-23. (canceled)
 24. A compound of formula R1-R2-R3, wherein: R1 is ahexacyclen; R3 is a β-nicotinamide adenine dinucleotide; and R2 is amoiety that links R1 and R3, wherein said moiety is a flexible linker.25. The compound of claim 24, wherein R2 is a polyglycine moietycontaining between 1 and 3 glycines.
 26. The compound of claim 24,wherein R2 is selected from the group consisting of polyethylene glycol(PEG), polystyrene-PEG, a [2-(2-aminoethoxy)ethoxy] acetic acid,allyloxycarbonyl-[2-(2-aminoethoxy)ethoxy] acetic acid,fluorenyl-methoxycarbonyl-[2-(2-aminoethoxy)ethoxy] acetic acid,ter-butyloxycarbonyl-[2-(2-aminoethoxy)ethoxy] acetic acid,benzyloxycarbonyl-[2-(2-aminoethoxy)ethoxy] acetic acid, and BMPS(N-(β-maleimido-propyloxy)succinimide).
 27. A compound of the formulaR1-R2-R3, wherein: R1 comprises a moiety that binds to a hemoglobinbinding site on a cytochrome b₅ at one or more amino acids selected fromthe group consisting of H26, E43, E44, E48, A54, D60, H80 and A88,wherein said moiety is a polyamine; R3 comprises a moiety that binds tothe cytochrome b₅ at one or more amino acids selected from the groupconsisting of I24, L25, H26 and H27, wherein said moiety is ATP or anATP analog; and R2 comprises a moiety that links R1 and R3, wherein saidmoiety is a flexible linker.
 28. The compound of claim 27, wherein thepolyamine is linear or cyclic.
 29. The compound of claim 27, wherein R1is hexacyclen.
 30. The compound of claim 27, wherein R3 isβ-nicotinamide adenine dinucleotide.
 31. The compound of claim 27,wherein R3 is 1,N6-ethenoadenosine 5′ triphosphate, β-nicotinamideadenine dinucleotide, 1,N6-ethenoadenosine hydrochloride,nicotinamide-1,6-ethenoadenosine, or coenzyme A.
 32. The compound ofclaim 27, wherein R1 is hexacyclen and R3 is β-nicotinamide adeninedinucleotide.
 33. The compound of claim 27, wherein R2 is a polyglycinemoiety containing between 1 and 3 glycines.
 34. The compound of claim27, wherein R2 is selected from the group consisting of polyethyleneglycol (PEG), polystyrene-PEG, [2-(2-aminoethoxy)ethoxy] acetic acid,allyloxycarbonyl-[2-(2-aminoethoxy)ethoxy] acetic acid,fluorenyl-methoxycarbonyl-[2-(2-aminoethoxy)ethoxy] acetic acid,ter-butyloxycarbonyl-[2-(2-aminoethoxy)ethoxy] acetic acid,benzyloxycarbonyl-[2-(2-aminoethoxy)ethoxy] acetic acid, and BMPS(N-(β-maleimido-propyloxy)succinimide).
 35. A pharmaceutical compositioncomprising the compound of claim 24 or a pharmaceutically acceptablesalt thereof.